Agencourt chloropure

Genomic DNA was extracted from clonally propagated blasticidin S‐resistant calli transformed with the T‐DNA monitoring vector using Agencourt ChloroPure (Beckman Coulter) according to the manufacturer's instructions. Suppression PCR was performed with adaptors and primers listed in Table S1 according to Nishizawa‐Yokoi et al. (2021 (link)). The junctions isolated by suppression PCR were confirmed by PCR using a T‐DNA‐specific primer paired with a primer specific to the plant genomic DNA near the insertion site.

Genomic DNA was extracted from regenerated shoots of tobacco, hygromycin-resistant rice calli, or regenerated rice plants, using Agencourt Chloro Pure (BECKMAN COULTER, USA), and target loci were amplified by PCR using the primer sets listed in supplemental table 1. For cleaved amplified polymorphic sequences (CAPS) analysis, PCR products were digested by the appropriate restriction enzymes, and then analyzed by agarose gel electrophoresis. A heteroduplex mobility assay (HMA) was performed using MultiNA (SHIMADZU, Japan) according to our previous report12 (link).

Phylogenetic analysis of the two Microcystis strains (NIES-4344 and NIES-4345) producing MC-FR and -WR variants was based on the ftsZ genotype. DNA was extracted from 10 mL of the culture using Agencourt ChloroPure (BECKMAN COULTER, Fullerton, CA, USA). The amount of DNA was measured using the Qubit BR Assay Kit (Thermo Fisher Scientific Inc., MA, USA). The first PCR was conducted using a set of primers, ftsF and ftsR [17 (link)], with overhang sequences of Illumina, respectively. The second PCR was conducted using NEBNext Multiplex Oligos for Illumina. Amplification of the gene was confirmed by agarose gel electrophoresis, and each single band was purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). The gel-extracted samples were purified twice using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The quality of the samples was verified by Agilent 2200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA, USA). DNA sequencing was performed using Illumina MiSeq (Illumina, San Diego, CA, USA) using the 600-cycle MiSeq Reagent Kit v3 (Illumina, San Diego, CA, USA).